Light-activated azobenzene peptide inhibitor of the PD-1/PD-L1 interaction

Inhibiting the PD-1/PD-L1 protein–protein interaction is a key immunotherapy for cancer. Antibodies dominate the clinical space but are costly, with limited applicability and immune side effects. We developed a photo-controlled azobenzene peptide that selectively inhibits the PD-1/PD-L1 interaction when in the cis isomer only. Activity is demonstrated in in vitro and cellular assays.


General procedure for peptide synthesis
The twelve amino acid CLP003 peptide (Ac-WHFSYNWRWLPP-NH 2 ) and the azobenzene substituted peptides (1-6) were synthesised on TentaGel S RAM resin (resin loading 0.23 mmol/g) using an automated peptide synthesiser. 1The AMPP unit was coupled identically as other amino acids.Tentagel S Ram resin (100 mg, 0.023 mmol) was suspended in DMF (2 mL) and allowed to swell for 20 minutes.The DMF was drained from the peptide vessel and Fmoc deprotection was carried out by addition of 20% piperidine in DMF (2 mL), which was shaken for 10 minutes, the repeated.This was removed and the resin was washed with DMF (3 x 2 mL).The resin was then treated with a solution of Fmoc-Pro-OH (4 equiv.compared to resin loading), to which HBTU (3.9 equiv.)and HOBt (4 equiv.)and DIPEA (8 equiv.) in DMF were added.The mixture was then shaken for 45 min.The vessel was drained and the resin washed with DMF (3 x 2 mL).The coupling reaction was then repeated followed by Fmoc deprotection (2 mL 20% piperidine in DMF, 10 min, twice) and finally the resin was washed with DMF.
Subsequent amino acids were coupled in an identical fashion.After the final amino acid coupling reaction (tryptophan) and Fmoc deprotection, the resin was treated with acetic anhydride (4 equiv.)and DIPEA (3.9 equiv.) in DMF and reacted for 45 minutes.The resin was washed with DCM (x 3) and MeOH (x 3) to remove any residual DMF.

CLP003 and alanine substituted peptides
The general procedure above was followed.Once synthesised the peptide was cleaved from the resin using 94:2.5:2.5 TFA:H 2 O:TIPS (5 mL) and shaken for three hours.The resulting solution was collected and the resin was washed with TFA (3 x 1 mL), the solutions were combined and concentrated under vacuum.The peptide was precipitated using cold diethyl ether and filtered.The peptide was purified using preparative RP-HPLC and concentrated under vacuum to yield a white solid (3.6 mg, 2.1 µmol, 9% This was repeated for the twelve alanine scanned peptides.Analytical RP-HPLC was performed using an Agilent 1200 HPLC, fitted with an Agilent ZORBAX Eclipse XDB-C8 column (4.6 x 150 mm, 5 μm) and a flow rate of 1 mL/min.Spectra were run with a solvent   Figure 6 a) The analytical HPLC traces of 6 reading at 214 nm top: after irradiation at 365 nm for 20 minutes showing predominantly the cis isomer (74:26) bottom: after being exposed for 20 minutes of ambient light showing predominantly the trans isomer (78:28) (Agilent eclipse XDB-C18 column (4.6 x 150 mm, 5 μm) and a flow rate of 1 mL/min.Spectra were run with a solvent gradient of 0-100% B over 20 min.Solvent A: H 2 O, 0.05% TFA, solvent B: MeCN, 0.05% TFA).B) UV/Vis spectra for 6 after being irradiated for 20 minutes and being kept in the dark while taking time interval readings showing that after 24 hours 6 remains predominantly in the cis isomer.

Thermal stability of 1
The peptide was dissolved in IMDM (ThermoFisher) with 10% FBS, Pen-Strep (100 µg/mL) and Normocin (100 µg/mL) to 1 mg/mL and incubated at 37 °C for 8 hours.The analytical HPLC trace was recorded every hour, with no observable change.

PD-1/PD-L1 HTRF binding assay
This assay was purchased from CisBio and was performed following the manufacturer's The emission ratio was calculated for each well was calculated with the following equation: The "specific signal" (ΔR) is obtained by subtracting the background from the signal of each

Figure 2 a
Figure 2 a) The analytical HPLC traces of 2 reading at 214 nm top: after irradiation at 365 nm for 20 minutes showing predominantly the cis isomer (71:29) bottom: after being exposed for 20 minutes of ambient light showing predominantly the trans isomer (70:30) (Agilent eclipse XDB-C18 column (4.6 x 150 mm, 5 μm) and a flow rate of 1 mL/min.Spectra were run with a solvent gradient of 0-100% B over 20 min.Solvent A: H 2 O, 0.05% TFA, solvent B: MeCN, 0.05% TFA).B) UV/Vis spectra for 2 after being irradiated for 20 minutes and being kept in the dark while taking time interval readings showing that after 24 hours 2 remains predominantly in the cis isomer.

3 Figure 3 a
Figure 3 a) The analytical HPLC traces of 3 reading at 214 nm top: after irradiation at 365 nm for 20 minutes showing predominantly the cis isomer (94:6) bottom: after being exposed for 20 minutes of ambient light showing predominantly the trans isomer (91:9) (Agilent eclipse XDB-C18 column (4.6 x 150 mm, 5 μm) and a flow rate of 1 mL/min.Spectra were run with a solvent gradient of 0-100% B over 20 min.Solvent A: H 2 O, 0.05% TFA, solvent B: MeCN, 0.05% TFA).B) UV/Vis spectra for 3 after being irradiated for 20 minutes and being kept in the dark while taking time interval readings showing that after 24 hours 3 remains predominantly in the cis isomer.

Figure 4 a
Figure 4 a) The analytical HPLC traces of 4 reading at 214 nm top: after irradiation at 365 nm for 20 minutes showing predominantly the cis isomer (80:20) bottom: after being exposed for 20 minutes of ambient light showing predominantly the trans isomer (82:18) (Agilent eclipse XDB-C18 column (4.6 x 150 mm, 5 μm) and a flow rate of 1 mL/min.Spectra were run with a solvent gradient of 0-100% B over 20 min.Solvent A: H 2 O, 0.05% TFA, solvent B: MeCN, 0.05% TFA).B) UV/Vis spectra for 4 after being irradiated for 20 minutes and being kept in the dark while taking time interval readings showing that after 24 hours 4 remains predominantly in the cis isomer.
protocol.Tag1-PD-L1 and Tag2-PD-1 was diluted 40-fold from the stock solution with PPI-Europium detection buffer.Anti-Tag1 Eu Cryptate reagent and Anti-Tag2 XL665 antibody was diluted 50-fold from the stock solution with PPI-Europium detection buffer.HTRF 96well low volume white plate was used.The samples were made to the desired concentration and 2 µL was pipetted into the well.The 4 µL of Tag-1-PD-L1 solution (25 nM) was added, followed by 4 µL of Tag2-PD-1 solution (250 nM).1X Anti-Tag1 Eu Cryptate reagent solution (25 nM|) and 1X Anti-Tag2 XL665 antibody solution (250 nM) were pre-mixed prior to dispensing 10 µL into the well, for a total volume of 20 µL.The plate was sealed and incubated for 1 hour at room temperature.Samples were analysed in triplicate.Once incubated, the plate was read using a CLARIOstar (BMG Labtech) microplate reader following manufacturer's protocols for time resolved fluorescence (excitation filter EX-TR, emission filters 620 (10) nm, 665 (10) nm, dichoric mirror LP-TR, integration delay (lag time) 60 µs, integration time 400 µs, number of flashes 200, gain for white plate 2500).